Journal: Nature Communications

Article Title: Alleviation of liver fibrosis by inhibiting a non-canonical ATF4-regulated enhancer program in hepatic stellate cells

doi: 10.1038/s41467-024-55738-1

Figure Lengend Snippet: a–c RNA-seq was conducted on the HMLE cells transduced with a scramble shRNA (shCtrl) or an ATF4-targeted shRNA (shATF4) and treated with indicated conditions (Twist-OE, Twist overexpression; Tg, thapsigargin). a A heatmap showing the expression of genes that were downregulated by ATF4 knockdown and ranked in the top 2.5% under Twist-OE or Tg treatment, respectively. b GO (gene ontology) enrichment analysis for genes downregulated by ATF4 knockdown for more than 1.5-fold in Tg treatment. A list of top enriched genesets (upper panel) and a GSEA (gene set enrichment analysis) plot showing the enrichment of the unfolded protein response pathway (lower panel) were shown. Hypergeometric test was used to identify significant enrichment pathways ( P < 0.01). The statistical significance (nominal P value) of the normalized enrichment score (NES) was generated by employing an empirical phenotype-based permutation test. c GO enrichment analysis for genes downregulated by ATF4 knockdown for more than 1.5-fold when Twist was induced. A list of top enriched genesets (upper panel) and a GSEA plot showing the enrichment of the epithelial-mesenchymal transition pathway (lower panel) were shown. Hypergeometric test was used to identify significant enrichment pathways ( P < 0.01). The statistical significance (nominal P value) of the normalized enrichment score (NES) was generated by employing an empirical phenotype-based permutation test procedure. d–f RNA-seq was conducted on the LX-2 cells transduced with a scramble shRNA (shCtrl) or an ATF4-targeted shRNA (shATF4) and treated with indicated conditions. Similar analyses were conducted as in ( a – c ). Hypergeometric distribution tests were applied for GO enrichment analysis and weighted Kolmogorov–Smirnov tests were applied for GSEA. These analyses were conducted on the average data of two biological replicates.

Article Snippet: Gene ontology (GO) and gene set enrichment analysis (GSEA) were conducted using the hallmark signature genesets from the Molecular Signature Database (MSigDB, Broad Institute), with an additional geneset “Amino Acid Deprivation” comprising the top 200 genes induced by an aminopeptidase inhibitor, CHR-2797, to mimic the condition of amino acid deprivation .

Techniques: RNA Sequencing, Transduction, shRNA, Over Expression, Expressing, Knockdown, Generated

Journal: Nature Communications

Article Title: Alleviation of liver fibrosis by inhibiting a non-canonical ATF4-regulated enhancer program in hepatic stellate cells

doi: 10.1038/s41467-024-55738-1

Figure Lengend Snippet: a 8-week-old male C57BL/6 mice were administered with CCl 4 (0.5 ml/kg) or solvent control (corn oil) and simultaneously with or without ISRIB (5 mg/kg) twice weekly for 5 weeks. n = 5–7 mice per group. Western blots showing the expression of EMT and fibrotic marker genes in the liver tissues of mice with different treatments. The experiment was repeated three times. b qPCR analysis showing the expression of fibrotic genes in hepatocytes and HSCs isolated from the liver tissues from ( a ) ( n = 3 biological replicates). c The representative images of Sirius red staining for liver tissues of ( a ) ( n = 6 images derived from 3 mouse livers per group). d The hepatic collagen content of the liver tissues of ( a ) was determined by hydroxyproline quantification ( n = 4 biological replicates). e 8-week-old male C57BL/6 mice underwent a sham (Sham) operation or surgery of common bile duct ligation (BDL), followed by biweekly administration of ISRIB (5 mg/kg) or solvent control for 4 weeks ( n = 5 mice per group). Western blots showing the expression of EMT and fibrotic marker genes in the liver tissues of mice with different treatments. The experiment was repeated three times. f 8-week-old male C57BL/6 mice were administered with solvent control, TAA (150 mg/kg), or a combination of TAA and ISRIB (5 mg/kg) twice weekly for 5 weeks ( n = 5 mice per group). Western blots showing the expression of EMT and fibrotic marker genes in the liver tissues of mice with different treatments. The experiment was repeated three times. g The hepatic collagen content of the liver tissues of e was determined by hydroxyproline quantification ( n = 4 samples per group). h The hepatic collagen content of the liver tissues of ( f ) was determined by hydroxyproline quantification ( n = 5 samples per group). i t-SNE representation of scRNA-seq analysis of HSCs from human liver of healthy donors ( n = 5) and cirrhotic patients ( n = 5). Cells from healthy or cirrhotic samples are highlighted in cyan or red, respectively. (adapted from GSE136103). j Violin plot showing the mRNA expression of ATF4 in HSCs of ( i ). k GSEA was performed with two genesets of ATF4 targets in a data set where genes were ordered by the fold-change of their expression comparing the HSCs of the cirrhotic patients and the healthy donors of ( i ). The geneset “ATF4 targets under TGFβ” (left) refers to genes regulated by ATF4 under TGFβ treatment in the LX-2 cells originating from the above bulk RNA-seq; The geneset “ATF4 targets under Tg” (right) refers to genes regulated by ATF4 under Tg treatment in the LX-2 cells also originated from the above bulk RNA-seq. The statistical significance (nominal P value) of the normalized enrichment score (NES) was generated by employing an empirical phenotype-based permutation test. l Spearman correlation of the Fib-4 scores and the expression of ATF4 in HSCs in the liver tissue from 15 cirrhotic patients. Determined by the intensity of IHC staining, the ATF4 expression in HSCs in these tissues was put into four categories: negative (1), low (2), medium (3), and high (4). Data are presented as mean ± s.e.m. Unpaired, two-tailed Student’s t tests were applied for ( b , d , g , h , j ). Kolmogorov–Smirnov tests were applied for ( k ). Spearman’s correlation was applied for ( l ). Source data are provided as a Source Data file.

Article Snippet: Gene ontology (GO) and gene set enrichment analysis (GSEA) were conducted using the hallmark signature genesets from the Molecular Signature Database (MSigDB, Broad Institute), with an additional geneset “Amino Acid Deprivation” comprising the top 200 genes induced by an aminopeptidase inhibitor, CHR-2797, to mimic the condition of amino acid deprivation .

Techniques: Solvent, Control, Western Blot, Expressing, Marker, Isolation, Staining, Derivative Assay, Ligation, RNA Sequencing, Generated, Immunohistochemistry, Two Tailed Test